Virus like particle purification

ABSTRACT

Methods for purifying human Calciviruses are disclosed, including Noroviruses and Sapoviruses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 60/906,821, filed Mar. 14, 2007.

STATEMENT OF GOVERNMENT SUPPORT

This invention was produced with government support from the US ArmyMedical Research and Material Command, under contract numbersDAMD17-01-C-0400 and W81XWH-05-C-0135. The government may have certainrights to the invention.

FIELD OF THE INVENTION

This application relates to methods for extracting and purifying viruslike particles (VLPs) from biological sources. More particularly, itrelates to methods for producing commercial grade VLPs at large scale.The methods employ a plurality of purification steps that yield purifiedVLPs.

BACKGROUND OF THE INVENTION

The human Caliciviruses Norovirus and Sapovirus are leading causes ofacute, non-bacterial gastroenteritis. In contrast to Norovirus,Sapovirus is known to give infections mainly in infants and youngchildren, however Sapovirus is increasingly found in the adultpopulations as well (Johansson et al., 2005, A nosocomialsapovirus-associated outbreak of gastroenteritis in adults. Scand JInfect Dis. 37(3):200-4). Noroviruses are non-cultivatable humanCaliciviruses that have emerged as the single most important cause ofepidemic outbreaks of nonbacterial gastroenteritis (Hardy, 1999, ClinLab Med. 19(3):675-90). The clinical significance of Noroviruses wasunder-appreciated prior to the development of sensitive moleculardiagnostic assays. The cloning of the prototype genogroup I Norwalkvirus (NV) genome and the production of virus-like particles (VLPs) froma recombinant Baculovirus expression system led to the development ofassays that revealed widespread Norovirus infections (Jiang et al.Norwalk Virus Genome Cloning and Characterization. Science 1990; 250:1580-1583; Jiang et al. 1992, J. Virol. 66(11):6527-32).

Noroviruses and Sapoviruses are single-stranded, positive sense RNAviruses that contain a non-segmented RNA genome. The viral genomeencodes three open reading frames, of which the latter two specify theproduction of the major capsid protein and a minor structural protein,respectively (Glass et al., The Epidemiology of Enteric Calicivirusesfrom Human: A Reassessment Using New Diagnostics. J Infect Dis 2000; 181(Sup 2): S254-S261). When expressed at high levels in eukaryoticexpression systems, the capsid protein of NV, and certain otherNoroviruses and Sapoviruses, self-assembles into VLPs that structurallymimic native Norovirus virions. When viewed by transmission electronmicroscopy, the VLPs are morphologically indistinguishable frominfectious virions isolated from human stool samples.

Although Norovirus and Sapovirus cannot be cultivated in vitro, due tothe availability of VLPs and their ability to be produced in largequantities, considerable progress has been made in defining theantigenic and structural topography of the Norovirus capsid. VLPspreserve the authentic conformation of the viral capsid protein whilelacking the infectious genetic material. Consequently, VLPs mimic thefunctional interactions of the virus with cellular receptors, therebyeliciting a strong host immune response while lacking the ability toreproduce or cause infection. In conjunction with the NIH, BaylorCollege of Medicine studied the humoral, mucosal and cellular immuneresponses to Norovirus VLPs in human volunteers in an academic,investigator-sponsored Phase I clinical trial. Orally administered VLPswere safe and immunogenic in healthy adults (Ball et al. 1999; Tacket etal. 2003). At other academic centers, preclinical experiments in animalmodels have demonstrated enhancement of immune responses to VLPs whenadministered intranasally with bacterial exotoxin adjuvants (Guerrero etal. 2001, Recombinant Norwalk Virus-like Particles AdministeredIntranasally to Mice Induce Systemic and Mucosal (Fecal and Vaginal)Immune Responses. J Vivol 2001; 75: 9713; Nicollier-Jamot et al. 2004,Recombinant Virus-like Particles of a Norovirus (Genogroup II Strain)Administered Intranasally and Orally with Mucosal Adjuvants LT andLT(R192G) in BALB/c Mice Induce Specific Humoral and CellularTh1/Th2-like Immune Responses. Vaccine 2004; 22:1079-1086; Periwal etal. 2003, Enhances Systemic and Mucosal Immune Responses to RecombinantNorwalk Virus-like Particle Vaccine. Vaccine 2003; 21: 376-385).

Small-scale methods for purifying Norovirus VLPs have been described inthe literature. For example, Norwalk virus VLP purification byultracentrifugation has been described (Jiang et al. 1990; 1992) and iscommonly employed by the Norovirus investigators in the field. However,while VLPs purified by ultracentrifugation have been used in humanclinical trials, the method is not suitable for producing commercialscale quantities of Calicivirus VLPs. Consequently, there remains a needto provide a scalable and efficient purification system capable ofpurifying VLPs from various biological sources.

SUMMARY OF THE INVENTION

Applicants have solved the need for scalable purification systems forCalicivirus VLPs by developing suitable chromatographic methods for theefficient purification of Calicivirus VLPs. The methods of the inventionare amenable to scaling for commercial production of purified VLPs.Thus, the present invention relates to methods of purifying Calicivirusvirus-like particles (VLPs) using chromatographic processes. Thechromatographic process may utilize more than one chromatographicmaterial and more than one mobile phase condition. The chromatographicmaterials and mobile phase conditions may be of different physical orchemical properties, making the chromatographic process orthogonal.

In some embodiments, the chromatographic materials and mobile phaseconditions are selected to retain VLPs. In other embodiments, thechromatographic materials and mobile phase conditions are selected topass through VLPs. In still other embodiments, the chromatographicmaterials and mobile phase conditions are selected to retaincontaminants in VLP preparations. In yet other embodiments, thechromatographic materials and mobile phase conditions are selected topass through contaminants in VLP preparations.

In some embodiments, among others, the chromatographic process of theinvention is a multistep chromatographic process employing two or morechromatographic steps. The sequence of the multistep chromatographicprocess of the present invention may be designed to produce VLPs meetingpreset specifications. For example, the purification method of thepresent invention may be used to purify VLPs to greater than about 70%,80%, 90%, 95%, or greater than 99% purity.

In one embodiment, the sequence of the multistep chromatographic processis designed to control the resulting composition of VLPs. In anotherembodiment, the sequence of the multistep chromatographic process isdesigned to reduce contaminant levels to levels considered acceptable byregulatory agencies for pharmaceutical grade drug substance. Forinstance, the contaminant level of the host cell DNA content may bereduced to less than 1%. The contaminant level of the host cell proteincontent may be reduced to less than 5%. The sequence of the multistepchromatographic process is designed to make VLPs consistent with cGMPregulations and suitable for pharmaceutical testing in humans.

The present invention also encompasses a method of contacting a solutioncontaining VLPs with a chromatographic material. In this regard, a celllysate containing VLPs may be contacted with the chromatographicmaterial wherein the cell lysate is filtered or purified byprecipitation prior to contact with the chromatographic material. Thesolution or cell lysate may be centrifuged without a sucrose gradient.In one embodiment, a clarified solution containing VLPs is contactedwith the chromatographic material. Alternatively, a VLP containingsolution from one chromatographic step is contacted with anotherchromatographic material.

The VLP containing solution may be produced using recombinantmethodologies. For example, the VLPs and VLP proteins may be produced inbacterial cells, insect cells, yeast cells, or mammalian cells.

In one embodiment, the present invention provides chromatographicmaterial comprising chromatographic resin in solution, chromatographicresin in a column or chromatographic functionality incorporated into amembrane or onto a surface. The chromatographic material may be designedfor the purification of proteins or nucleic acids. The chromatographicmaterial may further comprise ion-exchange, affinity, hydrophobicinteraction, mixed mode, reversed phase, size exclusion, and adsorptionmaterials. The adsorption material may be a resin or membrane.

In one embodiment, the chromatographic material comprises a calciumphosphate based material. The calcium phosphate based material may behydroxyapatite.

In another embodiment, the chromatographic material comprises an ionexchanger. The ion exchanger is a cation exchanger wherein the cationexchanger comprises sulfate, phosphate and carboxylate derivitizedchromatographic materials. In another embodiment, the ion exchanger isan anion exchanger, wherein the ion exchanger comprises positivelycharged chromatographic material. The positively charged chromatographicmaterial may be quaternary amine (Q) or diethylaminoethane (DEAE).

In yet another embodiment, the chromatographic material comprises ahydrophobic interaction material. The hydrophobic interaction materialmay comprise one or more functional groups selected from the groupconsisting of methyl, ethyl, t-butyl and phenyl. In one embodiment, thehydrophobic interaction chromatographic material is a methyl HIC resin.

In still another embodiment, the chromatographic material comprises areverse phase material. The reversed phase material comprises C2, C4, C8or C18 functionality. In another embodiment, the chromatographicmaterial comprises an affinity chromatographic material. The affinitychromatographic material comprises antibodies, dry resins, and metals.The dry resin may be cibachrom blue or polymixin.

In one embodiment, the chromatographic material comprises a sizeexclusion material wherein the size exclusion material is a resin ormembrane. The resin or membrane comprises pores of the same or differentsizes.

The present invention also provides a method of purifying VLPs whereinthe VLP-containing solution is adjusted to cause the retention of VLPs,with contaminating materials passing through the chromatographicmaterial. In some embodiments, the pH of the VLP-containing solution maybe adjusted with a buffer to more acidic values (e.g. pH less than 7).In other embodiments, the pH of the VLP-containing solution can beadjusted to more basic values (e.g. pH greater than 7). The buffer maycomprise phosphate, carboxylate, sulfate, acetate, citrate, tris or bistris. The buffer concentration may be in the range of about 10 to 1000mM.

In one embodiment, the ionic strength of the VLP containing solution isadjusted. The ionic strength may be adjusted by anions and cations fromthe Hofineister series such as ammonium sulfate. The ammonium sulfateconcentration may be greater than about 100 milli molar, or about 1, 2,or 2.4 molar.

In another embodiment, the ionic strength is adjusted by the addition ofa phosphate salt. In some embodiments, the phosphate salt is sodiumphosphate. The sodium phosphate concentration may be in the range ofabout 10 to 500 mM. In one embodiment, the sodium phosphateconcentration is about 100 mM.

In one embodiment of the invention, the pH of the chromatographicmaterial is adjusted prior to VLP application by equilibration with abuffer to cause the retention of VLPs. The pH may be adjusted to acidicvalues (e.g. less than 7), basic values (e.g. greater than 7), orneutral values (e.g. equal to 7). The equilibration buffer may comprisephosphate, carboxylate, sulfate, acetate, citrate, tris or bis tris.

In another embodiment, the ionic strength of the chromatographicmaterial is adjusted to cause retention of the VLPs. The adjustment maybe achieved by the addition of salt. The salt may comprise cations andanions from the Hofineister series such as ammonium sulfate. Theconcentration of ammonium sulfate may be greater than about 1, 2, or 2.4molar.

In another embodiment, the ionic strength of the chromatographicmaterial is adjusted by the addition of a phosphate salt. In someembodiments, the phosphate salt may be sodium phosphate. The sodiumphosphate concentration may be in the range of about 10 to 500 mM. Inone embodiment, the sodium phosphate concentration is about 100 mM.

In yet another embodiment, the organic solvent concentration of the VLPcontaining solution is adjusted to cause retention of VLPs.

The present invention further provides methods of purifying VLPs byselecting VLP containing solutions and chromatographic materials tocause retention of contaminating materials with the VLPs passing throughthe chromatographic resin. In so doing, the pH of the VLP containingsolution may be adjusted with a buffer, e.g., the pH adjusted to lessthan 7 or greater than or equal to 7. The buffer may comprise phosphate,carboxylate, sulfate, acetate, citrate, tris or bis tris. In oneembodiment, the buffer concentration is in the range of about 10 to 1000mM.

In one embodiment, the ionic strength of the VLP containing solution isadjusted to cause retention of contaminating materials with the VLPspassing through the chromatographic material. This may be achieved bythe addition of salt wherein the salt may comprise cations and anionsfrom the Hofineister series such as sodium phosphate. The sodiumphosphate concentration may be in the range of about 10 to 500 mM. Inone embodiment, the sodium phosphate concentration is about 100 mM.

In another embodiment, the pH of the chromatographic material isadjusted prior to VLP application by equilibration with a buffer tocause retention of contaminating materials with the VLPs passing throughthe chromatographic resin. The pH may be adjusted to less than 7 orgreater than or equal to 7 with a buffer that may comprise phosphate,carboxylate, or sulfate.

In another embodiment, the ionic strength of the chromatographicmaterial is adjusted prior to VLP application by equilibration with abuffer to cause retention of contaminating materials with the VLPspassing through the chromatographic resin. The ionic strength isadjusted by the addition of salt. The salt comprises cations and anionsfrom the Hofineister series such as sodium phosphate. In one embodiment,the sodium phosphate concentration is greater than about 10 mM.

In yet another embodiment, binding and elution of VLPs is controlled bythe amount of organic solvent present in the mobile phase. The organicsolvent concentration of the VLP containing solution may also beadjusted to cause retention of contaminating materials. The organicsolvent can be an alcohol such as methanol, ethanol or propanol or otherwater miscible organic solvents such as acetonitrile.

The present invention further provides methods of purifying VLPs fromCalicivirus virus-like particles (VLPs) such as Norovirus and SapovirusVLPs. The Norovirus comprises Genogroup I, Genogroup II, Genogroup III,and Genogroup IV Noroviruses. Sapovirus comprises five Genogroups (1-V),among which only Genogroups I, II, IV, and V are known to infect humans(Farkas et al. 2004, Genetic diversity among sapoviruses. Arch Virol.2004; 149:1309-23).

The present invention further provides a pharmaceutical agent preparedby the multistep chromatographic process. The pharmaceutical agent maybe a vaccine such as a Norovirus vaccine.

The present invention further provides an analytical reagent prepared bythe multistep chromatographic process described herein. The analyticalreagent may be purified to a desired purification level, which can beused in a diagnostic assay or kit.

These and other embodiments of the invention will become apparent upon afull consideration of the invention presented below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an example gel showing changes in VLP purity as a function ofchromatographic step.

FIG. 2 is a SDS-PAGE gel/Coomassie Stain of HydroxyapatiteChromatography Column Fractions.

FIG. 3 is a SDS-PAGE gel/Coomassie Stain of HIC Chromatography ColumnFractions.

FIG. 4 is a SDS-PAGE gel/Coomassie Stain of DEAE Chromatography ColumnFractions.

FIG. 5 is a SDS-PAGE gel/Coomassie Stain of Diafiltraion Fractions.

FIG. 6 is an image of transmission Electron Micrograph of Norwalk virusVLPs purified chromatographically. Particles are approximately 34 to 38nM range in size.

FIG. 7 is an image of transmission Electron Micrograph of Norwalk virusVLPs purified by ultracentrifugation. These particles are also 34 to 38nM in size.

FIG. 8 is a graph showing CD spectra of column-purified VLPs at 10° C.(dash line) and 90° C. (solid line) as a function of pH.

FIG. 9 is a graph showing CD signal of column-purified VLPs monitored at205 nm as a function of temperature and pH.

FIG. 10 is a graph showing CD signal of column-purified VLPs monitoredat 222 nm as a function of temperature and pH.

FIG. 11 is a graph showing CD spectra at 10° C. (dash line) and 90° C.(solid line) as a function of pH, of VLPs purified byultracentrifugation.

FIG. 12 is a graph showing CD spectra at 205 nm as a function oftemperature and pH of VLPs purified by ultracentrifugation.

FIG. 13 is a graph showing CD signal at 222 nm as a function oftemperature and pH, of VLPs purified by ultracentrifugation.

FIG. 14 is a chromatogram from the Cation Exchange purification stepused for Houston virus VLPs.

FIG. 15 is a SDS-PAGE gel/Coomassie Stain of Cation Exchange fractionsof Houston virus VLPs.

FIG. 16 is a chromatogram from Methyl HIC Chromatography purificationstep used for Houston virus VLPs.

FIG. 17 is a SDS-PAGE gel/Coomassie Stain of Methyl HIC fractions ofHouston virus VLPs.

FIG. 18 is a SDS-PAGE gel/Coomassie Stain of purified Houston virusprotein.

FIG. 19 is an HPLC-SEC chromatogram of purified Houston virus protein.

FIG. 20. Silver stained SDS-PAGE gel showing purification of a HoustonVLP preparation with a 20% ammonium sulfate precipitation. “Ammsuspension” is the initial ammonium sulfate suspension. “Amm Supt” isthe supernatant which results when the ammonium sulfate suspension iscentrifuged. “Citrate supt” is the dissolved precipitated material. Ofinterest is the “amm supt” lane which highlights the amount ofnon-precipitated contaminant material.

FIG. 21. Graph comparing the components of the initial Houston VLPpreparation to the ammonium sulfate precipitated and redissolvedmaterial. Note the significant improvement in purity in the 20% ammoniumsulfate preparation by the decrease in percent host cell protein to VLP(HCP/VLP %).

FIG. 22. Silver stained SDS-PAGE gel depicting purification process ofHouston VLPs with precipitation followed by anion exchangechromatography. As shown by comparing lane 2 to lanes 5 and 6,precipitating the VLP with pH adjustment increases the purity. Lane 8illustrates the ability of column chromatography to concentrate theVLPs.

FIG. 23. Coomassie stained SDS-PAGE gel showing the purification processof Laurens VLPs with precipitation followed by anion exchangechromatography. A comparison of lanes 5 through 8 to lane 11 illustratesthe increase in purity of Laurens VLP samples obtained with capturechromatography.

FIG. 24. SE-HPLC analysis of GI Norovirus VLPs at various pH values.Panel A. SE-HPLC analysis of GI Norovirus VLPs at pH 2. The absorbancepeak at about 17 min corresponds to elution of intact, monodisperseVLPs. Panel B. SE-HPLC analysis of Norovirus GI VLPs at pH 8. Theabsorbance peak at about 16 min corresponds to elution of intact,monodisperse VLPs. Panel C. SE-HPLC analysis of Norovirus GI VLPs at pH8.5. The absorbance peak at about 33 min corresponds to elution of thestable intermediate fragment of the VLP. Chromatograms show absorbanceprofiles at 230 nm (upper) and 280 nm (lower).

FIG. 25. SE-HPLC analysis of GII Norovirus VLP at various pH levels.Panel A. SE-HPLC analysis of GII Norovirus VLPs at pH 2. The absorbancepeak at about 17 min corresponds to elution of intact, monodisperseVLPs. Panel B. SE-HPLC analysis of GII Norovirus VLPs at pH 9.5. Theabsorbance peak at about 17 min corresponds to elution of intact,monodisperse VLPs. Panel C. SE-HPLC analysis of GII Norovirus VLPs at pH10. The absorbance peak at about 34 min corresponds to elution of thestable intermediate fragment of the VLP. Chromatograms show absorbanceprofiles at 230 nm (upper) and 280 nm (lower).

DETAILED DESCRIPTION OF THE INVENTION Overview

All publications and patent applications cited throughout this patentare incorporated by reference to the same extent as if each individualpublication or patent/patent application is specifically andindividually indicated to be incorporated by reference in theirentirety.

Modifications and variations of this invention will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is not to be construedas limited thereby.

The present invention relates to methods for the purification ofCalicivirus virus-like particles (VLPs) including Norovirus VLPs andSapovirus VLPs. By “Norovirus,” “Norovirus (NOR),” “norovirus,” andgrammatical equivalents herein, are meant members of the genus Norovirusof the family Caliciviridae. In some embodiments, a Norovirus caninclude a group of related, positive-sense single-stranded RNA,nonenveloped viruses that can be infectious to human or non-humanmammalian species. In some embodiments, a Norovirus can cause acutegastroenteritis in humans. Noroviruses also can be referred to as smallround structured viruses (SRSVs) having a defined surface structure orragged edge when viewed by electron microscopy. Included within theNoroviruses are at least four genogroups (GI-IV) defined by nucleic acidand amino acid sequences, which comprise 15 genetic clusters. The majorgenogroups are GI and GII. GIII and GIV are proposed but generallyaccepted. Representative of GIII is the bovine, Jena strain. GIVcontains one virus, Alphatron, at this time. For a further descriptionof Noroviruses see Vinje et al. J. Clin. Micro. 41:1423-1433 (2003). By“Norovirus” also herein is meant recombinant Norovirus virus-likeparticles (rNOR VLPs). In some embodiments, recombinant expression of atleast the Norovirus capsid protein encoded by ORF2 in cells, e.g., froma baculovirus vector in Sf9 cells, can result in spontaneousself-assembly of the capsid protein into VLPs. In some embodiments,recombinant expression of at least the Norovirus proteins encoded byORF2 and ORF3 in cells, e.g., from a baculovirus vector in Sf9 cells,can result in spontaneous self-assembly of the capsid protein into VLPs.VLPs are structurally similar to Noroviruses but lack the viral RNAgenome and therefore are not infectious. Accordingly, “Norovirus”includes virions that can be infectious or non-infectious particles,which include defective and defective-interfering particles.

Non-limiting examples of Noroviruses include Norwalk virus (NV, GenBankM87661, NPo₅₆₈₂₁), Southampton virus (SHV, GenBank L07418), DesertShield virus (DSV, U04469), Hesse virus (HSV), Chiba virus (CHV, GenBankAB042808), Hawaii virus (HV, GenBank U0761 1), Snow Mountain virus (SMV,GenBank U70059), Toronto virus (TV, Leite et al., Arch. Virol.141:865-875), Bristol virus (BV), Jena virus (JV, AJ01099), Marylandvirus (MV, AY032605), Seto virus (SV, GenBank AB031013), Camberwell (CV,AF145896), Lordsdale virus (LV, GenBank X86557), Grimsby virus (GrV,AJ004864), Mexico virus (MXV, GenBank U22498), Boxer (AF538679), C59(AF435807), VA115 (AY038598), BUDS (AY660568), Houston virus (HoV,AY502023), MOH (AF397156), Parris Island (PiV; AY652979), VA387(AY038600), VA207 (AY038599), and Operation Iraqi Freedom (OIF,AY675554). Non-limiting examples of Sapoviruses include Sapporo virus(SV), Houston/86 [U95643] (Hu/SLV/Hou/1986/US), Houston/90 [U95644](Hu/SLV/Hou 27/1990/US), London 29845 [U95645] (Hu/SLV/Lon29845/1992/UK), Manchester virus [X86560] (Hu/SLV/Man/1993/UK),Parkville virus [U73124](Hu/SLV/Park/1994/US), Sapporo virus [U65427](Hu/SLV/SV/1982/JP). Additional viral strains of Caliciviruses continueto be identified and are contemplated for use in the methods of thepresent invention (ICTVdB—The Universal Virus Database, version 4.http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/). The nucleic acid andcorresponding amino acid sequences of each are all incorporated byreference in their entirety. In some embodiments, a cryptogram can beused for identification purposes and is organized: host species fromwhich the virus was isolated/genus abbreviation/speciesabbreviation/strain name/year of occurrence/country of origin. (Green etal., Human Caliciviruses, in Fields Virology Vol. 1 841-874 (Knipe andHowley, editors-in-chief, 4th ed., Lippincott Williams & Wilkins 2001)).Representative examples of purifying Norwalk virus VLPs and Houstonvirus VLPs are discussed herein.

By “VLP preparation” is intended any solution containing VLPs, and othermaterials that are sought to be purified. The VLP preparation can beproduced by a number of methods, including cultivation in a host cell invitro including any one of batch, perfusion, or cell factory methods, orin vivo in an appropriate animal host. In the former instance, virallyinfected cells can be harvested, separated from the growth media, andthe VLP protein either released into the media via a budding process orliberated by lysis of the cells and separation from cellular debris. Inthe latter instance, a tissue or organ harboring the virus can beremoved and the VLP proteins also liberated by lysis of cells thatcomprise the tissue or organ, and separated from cellular/tissue debris.

As used herein, “virus-like particle(s) or VLPs” refer to a virus-likeparticle(s), fragment(s), or portion(s) thereof produced from a capsidprotein coding sequence of Calicivirus and comprising antigeniccharacteristic(s) similar to those of infectious Calicivirus particles.VLPs can be any structural proteins wherein the structural proteins areencoded by one or more nucleic acid sequences. VLPs may includeindividual structural proteins, i.e., protein monomers, or dimers, orprotein complexes spontaneously formed upon purification of recombinantstructural proteins, i.e., self-assembling or intact VLPs, or aggregatedVLPs. VLPs may also be in the form of capsid monomers, protein orpeptide fragments of VLPs or capsid monomers, or aggregates or mixturesthereof. They may be produced using structural protein fragments ormutated forms thereof, e.g., structural proteins that have been modifiedby the addition, substitution or deletion of one or more amino acids.VLPs are morphologically and antigenically similar to authentic virions.VLPs may be produced in vivo, in suitable host cells, e.g., mammalian,yeast, bacterial, and insect host cells.

The present invention provides methods of large-scale purification ofrecombinant VLPs that were produced. The methods include preparing asolution, a cell lysate or culture supernatant from the host cell linethen passing the lysate or culture supernatant over various combinationsof chromatography materials or media. The host cell line may be culturedin Petri dishes, roller bottles, a bioreactor, or using anothertechnique suitable for large-scale cell culture.

A skilled practitioner of this art will appreciate that other virus likeparticles may be purified using the process of the instant invention byadapting certain of its features as are appropriate to the VLP beingpurified. Suitable VLPs are those readily purified using multiplechromatographic steps in the purification process, chromatographicmaterials such as hydroxyapatite, hydrophobic interaction, ion exchangeand size exclusion chromatographic materials.

A “bacterial cell” is herein defined to include prokaryotic cells thatmay be propagated in culture. The bacterial cell may act as a host cellfor the recombinant expression of heterologous proteins. The bacterialcell may be transformed, transfected or infected with a vector forexpression of a protein sequence inserted into the vector. Examples ofsuitable bacterial cells include, but are not limited to E. coli, B.megaterium, B. subtilis and B. brevis and various species ofCaulobacter, Staphylococcus, and Streptomyces.

A “yeast cell” is herein defined to include the group consisting ofsmall, unicellular organisms capable of growth and reproduction throughbudding or direct division (fission), or by growth as simple irregularfilaments (mycelium). The yeast cell may be transformed or transfectedwith a heterologous vector for expression of a nucleic acid sequenceinserted into the heterologous vector. An example of a yeast cellincludes Saccharomyces cerevisiae, commonly used for transfection andexpression of heterologous proteins.

A “mammalian cell culture” is herein defined to include the group ofcells derived from a mammalian source capable of surviving ex vivo in acell culture medium. The mammalian cell may be a primary cell, directlyderived from a mammalian cell source. More typically, the mammalian cellin a mammalian cell culture will be immortalized, i.e. capable of growthand division through an indeterminate number of passages or divisions.

An “insect cell” is herein defined to include the group of cells derivedfrom an insect source capable of surviving ex vivo from an insect host.The insect cell may be transformed, transfected or infected with aheterologous vector for expressions of a protein sequence inserted intothe heterologous vector. Examples of insect cells include High Five™cells, Aedes albopictus cells, Drosophila melanogaster cells, Sf9 insectcells and Mamestra brassicae cells.

“Lysis” refers to the process of opening virally infected cells bychemical, or physical means, or as part of the viral life cycle therebyallowing for the collection of VLPs.

By “porous chromatographic material” is meant virtually any type ofmaterial commonly used in the separation of molecules primarily based ontheir size, hydrophobicity and charge. As exemplified herein, “porouschromatographic material” includes dextran (e.g. Sephadex™ resins), orother porous materials that can be composed of a variety of materialsincluding agarose, poly-styrene divinyl-benzene, polymethacrylate,silica, aliphatic acrylic polymers (e.g. Amberlite™ resins), with avariety of surface derivitizations (e.g., hydrophilic, ionic,hydrophobic, etc.).

As used herein, the term “precipitation” refers to the adjustment ofsolution conditions through the addition or removal of salt, theaddition of organic solvent, the concentration of the protein containingsolution or the adjustment of pH resulting in either the selectiveprecipitation of molecules (VLPs or contaminant). Insoluble orprecipitated material is then separated from the soluble material usinga number of techniques such as centrifugation or filtration.

“Hydroxyapatite chromatography” refers to a method of purifying proteinsthat utilizes an insoluble hydroxylated calcium phosphateCa₁₀(PO₄)₆(OH)₂, which forms both the matrix and ligand. Functionalgroups consist of pairs of positively charged calcium ions (C-sites) andclusters of negatively charged phosphate groups (P-sites). Theinteractions between hydroxyapatite and proteins are complex andmulti-mode. In one method of interaction, however, positively chargedamino groups on proteins associate with the negatively charged P-sitesand protein carboxyl groups interact by coordination complexation toC-sites. Shepard, J. of Chromatography 891:93-98 (2000). Crystallinehydroxyapatite was the first type of hydroxyapatite used inchromatography, but it was limited by structural difficulties. Ceramichydroxyapatite (cHA) chromatography was developed to overcome some ofthe difficulties associated with crystalline hydroxyapatite, such aslimited flow rates. Ceramic hydroxyapatite has high durability, goodprotein binding capacity, and can be used at higher flow rates andpressures than crystalline hydroxyapatite. Vola et al., BioTechniques14:650-655 (1993).

By “size exclusion chromatography” is meant a method for separatingmolecules using porous chromatographic material. Size exclusionchromatography can consist of one or more distinct types of porouschromatographic material used in a single step, or one or more distincttypes of porous chromatographic material used in multiple separatesteps. As used herein, an example of “size exclusion chromatography”where more than one porous chromatographic material is used isAmberlite™ XAD7HP and Sephadex™ G-50.

An “affinity material” is a solid-state material bound to a substrate orligand, which in turn binds selectively to a protein of interest or aprotein attached to an affinity tag. Upon binding, the protein ofinterest is retained within the column or other purifying apparatus, andmay thus be separated from any impurities present in the VLPpreparation. After washing of the affinity matrix, the protein ofinterest, may be eluted from the column or other apparatus in asubstantially purified form. Examples of affinity matrices includechromatography medium, such as agarose, cellulose, Sepharose, Sephadexand other chromatography medium, polystyrene beads, magnetic beads,filters, membranes and other solid-state materials bound to ligands orsubstrates which bind to the affinity tag of choice.

As used herein, “to purify” a protein means to reduce to a given levelof purity the amounts of foreign or objectionable elements, especiallybiological macromolecules such as proteins or DNA, that may be presentin a sample of the protein. For instance, the methods of the presentinvention may be used to purify VLPs for greater than about 70%, 80%,90%, 95% or greater than 99% purity. The presence of foreign proteinsmay be assayed by any appropriate method including, but not limited togel electrophoresis and staining or western blot analysis, HPLC and/orELISA assay. The presence of DNA may be assayed by any appropriatemethod including gel electrophoresis and staining, DNA binding proteinsand/or assays employing polymerase chain reaction.

As used herein, the terms “chromatographic material,” “chromatographicmedium,” “chromatographic matrix,” and “chromatographic resin” and theirgrammatical equivalents are used interchangeably throughout thespecification.

Description of the Purification Procedure

The present invention relates to the purification of virus-likeparticles (VLP) from biological source materials. More specifically itrelates to the use of chromatographic methods as a means to removeimpurities and contaminants that may be detrimental to the recombinantVLP integrity or its subsequent use.

The disclosed invention contemplates using a single or plurality ofchromatographic step(s) in order to purify VLPs from a biologicalsource. Different chromatographic materials, used in varying orders andcombinations, are contemplated by the present invention. Thechromatographic step(s) may utilize more than one chromatographicmaterial and more than one mobile phase condition. The chromatographicmaterials and mobile phase conditions may be of different physical orchemical properties, and thus are orthogonal.

Chromatographic materials include, but are not limited to, ion-exchange,affinity, hydrophobic interaction, mixed mode, reversed phase, sizeexclusion, and adsorption materials. The invention also contemplatesmany support medium, including agarose, cellulose, silica, andpoly(stryrene-divinylbenzene) (PSDVB). In addition, multiplechromatographic methods can be used including conventionalchromatography, HPLC (High Performance Liquid Chromatography or Highpressure Liquid Chromatography), or perfusion chromatography. Oneskilled in the art will also appreciate that the size of the column(i.e., diameter and length) will depend on several factors such as thevolume of material to be loaded, the concentration of VLPs to bepurified, and the desired resolution or purity.

Cell Lysate Preparation

The practice of the invention employs techniques of molecular biology,protein analysis and microbiology, which are within the skills of apractitioner in the art. Such techniques are explained fully in, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley& Sons, New York, 1995.

VLPs can be purified from cell lysates prepared from a number ofbiological sources including cell lines, tissues, etc. Often VLPs willbe purified from a cell lysate preparation made from virus infectedcells, where the cells have been grown using cell culture methods. TheVLP containing cell lysates may be produced using recombinantmethodologies. For example, the VLPs and VLP proteins may be produced inbacterial cells., insect cells, yeast cells, or mammalian cells. Forexample, Norovirus VLPs can be isolated from Baculovirus-infected SF9cells, etc. Cells may be infected at high multiplicity of infection inorder to optimize yield.

Any method suitable for releasing VLP proteins from infected cells maybe utilized to prepare a cell lysate containing VLP. VLP protein mayalso be released into the growth media via a budding process. VLPs maybe recovered via separation from the media and cellular debris orreleased from infected cells using techniques known in the art. Themethods of lysing virally infected cells may include using hypotonicsolution, hypertonic solution, sonication, pressure, or a detergent. Inone embodiment, the technique is to use a detergent. In anotherembodiment, depending on the amount of DNA and RNA in the sample, thetechnique is to also use a nuclease in combination with a detergent.

Numerous detergents are available to solubilize cells, includingnon-ionic or ionic detergents. An enzymatic agent may be used to treatthe cell lysate consisting of one or more enzymes, preferably an RNAseand/or a DNAse, or a mixture of endonucleases as would be known to theordinarily skilled artisan. It is well known that nucleic acids mayadhere to cellular material which can interfere with the inventionchromatographic purification scheme by causing cellular or viralaggregation, resulting in little if any VLPs being recovered.

Clarification

Prior to the clarification step, the cell lysate preparation followingtreatment with detergent, or if preferred, detergent and nuclease, maybe treated to remove large particulate matter. This can be accomplishedby a number of procedures including low speed centrifugation, orfiltration. The type of filter or membrane used (i.e. composition andpore size) is within the knowledge of the skilled practitioner of theart to purify particular VLPs.

One embodiment of the invention involves clarification by precipitation.The desired VLP proteins may be recovered from the cell lysate orculture supernatant by the use of precipitation techniques well known tothose in the art, such as by the use of protein precipitation agentsincluding, but not exclusively, PEG, sodium sulfate, ammonium sulfate,glycine or temperature. The precipitation is preferably carried out withcarefully selected concentrations of the chemical agents as this reducesco-precipitation of contaminating proteins. Precipitated proteins arethen separated from soluble materials by filtration or bycentrifugation. In one embodiment of the invention, the VLPs areprecipitated by reducing the ionic strength of the solution through theaddition of deionized water. In another embodiment, VLPs areprecipitated by the addition of ammonium sulfate. The precipitated VLPsare then collected using a low speed centrifugation and resuspended inbuffer

Another embodiment of the invention may involve clarification usingporous chromatographic materials. In purifying VLP from certain celllysates and depending on the amount of cellular aggregates present, itmay be desirable to use a single porous chromatographic material toperform size exclusion chromatography. In these instances it may besufficient to employ a pre-clarification (i.e. filtration) step,followed by size exclusion chromatography using a single porouschromatographic material preferably made of dextran, and more preferablycertain Sephadex™ resins. Generally, a cell lysate or cell culturesupernatant, obtained by means that are well known in the art, issubject to clarification.

In one embodiment, the present invention provides chromatographicmaterial comprising chromatographic resin in solution, chromatographicresin in a column or chromatographic functionality incorporated into amembrane or onto a surface. By “membrane” is meant virtually any type ofmaterial commonly used in the separation of molecules primarily based ontheir size. As exemplified herein, “membrane' includes filters or otherporous materials that can be used for molecule separation.

The chromatographic material may be designed for the purification ofproteins or nucleic acids. The chromatographic material may furthercomprise ion-exchange, affinity, hydrophobic interaction, mixed mode,reversed phase, size exclusion, and adsorption materials. Exemplarychromatographic materials of the present invention are described in moredetail below. It should be understood that the chromatographic materialsare provided for the purpose of illustration only and the inventionshould in no way be construed as being limited to these chromatographicmaterials or steps but rather should be construed to encompass any andall chromatographic materials or steps that can be used for protein ornucleic acid purification in general and VLP purification in particular.

The chromatographic methods discussed below can be run as individualsteps, or sequentially, or in tandem. By “in tandem” is meant that aneluate from one chromatography is directly applied to the nextchromatography without an intervening eluate collection step.Alternatively, fractions of an eluate may be pooled and collected priorto being applied to the next chromatography.

Hydroxyapatite Chromatographic Material

In some embodiments, the chromatographic material comprises a calciumphosphate based material. The calcium phosphate based material may behydroxyapatite. Various hydroxyapatite chromatographic materials orresins are available commercially, and any available form of thematerial can be used in the practice of this invention. In oneembodiment of the invention, the hydroxyapatite is in a crystallineform. Hydroxyapatites for use in this invention may be those that areagglomerated to form particles and sintered at high temperatures into astable porous ceramic mass.

The particle size of the hydroxyapatite may vary widely, but a typicalparticle size ranges from 1 μm to 1,000 μm in diameter, and may be from10 μm to 100 μm. In one embodiment of the invention, the particle sizeis 20 μm. In another embodiment of the invention, the particle size is40 μm. In yet another embodiment of the invention, the particle size is80 μm.

This invention may be used with hydroxyapatite resin that is loose,packed in a column, or in a continuous annular chromatograph. In oneembodiment of the invention, ceramic hydroxyapatite resin is packed in acolumn. The choice of column dimensions can be determined by the skilledartisan. In one embodiment of the invention, a column diameter of atleast 0.5 cm with a bed height of about 20 cm may be used for smallscale purification. In an additional embodiment of the invention, acolumn diameter of from about 35 cm to about 60 cm may be used. In yetanother embodiment of the invention, a column diameter of from 60 cm to85 cm may be used.

Eluate from the hydroxyapatite column containing VLPs may be pooled andapplied to another chromatographic resin such as the hydrophobicinteraction chromatographic resin.

Hydrophobic Interaction Chromatographic Material

In some embodiments, the chromatographic material comprises ahydrophobic interaction material. The hydrophobic interaction materialmay comprise one or more functional groups selected from the groupconsisting of methyl, ethyl, t-butyl and phenyl.

Hydrophobic interaction chromatography (“HIC”) is a valuable techniquefor the separation of proteins under high salt conditions (see,generally, HPLC of Biological Macromolecules. Methods and Applications,Gooding, K. M. et al., Eds., Marcel Dekker, Inc. (1990)). With regard toproteins, HIC separation is based on the interactions of the hydrophobicamino acid residues of the protein with immobilized hydrophobic moietiesimmobilized to a chromatographic support. The immobilized hydrophobicmoieties may be selected from a broad range of alkyl and aryl groups.PEG is an immobilized moiety that is commonly used in HICchromatography. The hydrophobicity of the moiety increases withincreasing alkyl length. The protein is adsorbed to the column in highsalt (1-3M NH₄(SO₄)₂), and is eluted by lowering the ionic strength.Methods of conducting HIC are described by Cameron, G. W. et al. (Meth.Molec. Cell. Biol. 4:184-188 (1993)), Raymond, J. et al. (J. Chromatog.212:199-209 (1981)), Ochoa, J. I. (Biochimie 60:1-15 (1978)),Roggenbuck, D. et al. (J. Immunol. Meth. 167:207-218 (1994)),Michaelson, S. et al. (Pol. J. Food Nutr. Sci. 3/44:5-44 (1994), Rippel,G. et al. (J. Chromatog. 668:301-312 (1994)), Szepesy, L. et al. (J.Chromatog. 668:337-344 (1994)), Huddleston, J. G. et al. (Biotechnol.Bioeng. 44:626-635 (1994)), Watanabe, E. et al. (Annl. NY Acad. Sci.721:348-364 (1994)), all of which are herein incorporated by reference.

A variety of commercially available HIC column chemistries which span awide range of hydrophobicities should make it possible to find anappropriate ligand which allows for chromatographic separation. Forexample, HIC columns may be purchased from Synchrom and Bio-Rad(Hercules Calif.) covering the full range in available alkyl andaromatic ligands. In one embodiment, the HIC column used in theinvention is methyl HIC.

Size Exclusion Chromatographic Material

In some embodiments, the chromatographic material comprises a sizeexclusion material wherein the size exclusion material is a resin ormembrane. As intended herein, size-exclusion chromatography involvesseparating molecules primarily based on their size. The matrix used forsize exclusion is preferably an inert gel medium which can be acomposite of cross-linked polysaccharides, e.g., cross-linked agaroseand/or dextran in the form of spherical beads. The degree ofcross-linking determines the size of pores that are present in theswollen gel beads. Molecules greater than a certain size do not enterthe gel beads and thus move through the chromatographic bed the fastest.Smaller molecules, such as detergent, protein, DNA and the like, whichenter the gel beads to varying extent depending on their size and shape,are retarded in their passage through the bed. Molecules are thusgenerally eluted in the order of decreasing molecular size.

Porous chromatographic resins appropriate for size-exclusionchromatography of viruses may be made of dextran, and cross-linkeddextrans. Most commonly used are those under the tradename, “SEPHADEX”available from Amersham Biosciences. The type of SEPHADEX, or othersize-exclusion chromatographic resin used is a function of the type ofVLP sought to be purified, and the nature of the cell culture lysatecontaining the VLP. Other size exclusion supports from differentmaterials of construction are also appropriate, for example Toyopearl55F (polymethacrylate, from Tosoh Bioscience, Montgomery Pa.) andBio-Gel P-30 Fine (BioRad Laboratories, Hercules, Calif.).

For size exclusion chromatography a concentrated pool of partiallypurified VLPs are loaded onto a column containing an appropriatepreparative size exclusion chromatography column (such as a columncontaining Sephadex G200 or Superpose 6 resins) that had beenequilibrated in a suitable buffer (e.g., a phosphate buffer).

The present invention further provides chromatographic materialscomprising an ion exchanger. The ion exchanger may be a cation exchangerwherein the cation exchanger comprises sulfate, phosphate andcarboxylate derivitized chromatographic materials. The ion exchanger mayalso be an anion exchanger, wherein the anion exchanger comprisespositively charged chromatographic material. The positively chargedchromatographic material may be quaternary amine (Q) ordiethylaminoethane (DEAE).

Anion Exchange Chromatographic Material

Anion Exchange chromatography uses a positively-charged organic moietycovalently cross-linked to an inert polymeric backbone. The latter isused as a support for the resin. Representative organic moieties aredrawn from primary, secondary, tertiary and quaternary amino groups;such as trimethylaminoethyl (TMAE), diethylaminoethyl (DEAE),dimethylaminoethyl (DMAE), and other groups such as thepolyethyleneimine (PEI) that already have, or will have, a formalpositive charge within the pH range of approximately 5 to approximately9.

In one embodiment, an anion exchange resin consisting of DMAE, TMAE,DEAE, or quaternary ammonium groups is used. A number of anion exchangeresins sold under the tradename Fractogel (Novagen) use TMAE, DEAE, DMAEas the positively-charged moiety, and a methacrylate co-polymerbackground. Resins that use quaternary ammonium resins and quaternaryammonium resins of the type sold under the trade name Q SOURCE-30(Amersham Biosciences) may also be employed. Q SOURCE-30 has a supportmade of polystyrene cross-linked with divinylbenzene.

Several possible anion exchange media are known that can be used in suchcolumns including N-charged amino or imino resins such as POROS 50 PI™,Q SEPHAROSE™, any DEAE, TMAE, tertiary or quaternary amine, or PEI-basedresin One skilled in the art will appreciate that recombinant VLPs canbe purified on an anion exchange column either before or afterpurification on other columns.

The anion-exchange chromatographic resin, can be used in gravity columnchromatography or high pressure liquid chromatography apparatus usingradial or axial flow, fluidized bed columns, or in a slurry, that is,batch, method. In the latter method, the resin is separated from thesample by decanting or centrifugation or filtration or a combination ofmethods.

The principle of ion-exchange chromatography is that charged moleculesadsorb to ion exchangers reversibly so that molecules can be bound oreluted by changing the ionic environment. Separation on ion exchangersis usually accomplished in two stages: first, the substance to beseparated is bound to the exchanger, using conditions that give stableand tight binding; then the substance is eluted with buffers ofdifferent pH, or ionic strength, depending on the properties of thesubstance being purified.

More specifically, and as applied to the instant invention, the basicprinciple of ion-exchange chromatography is that the affinity of a VLPfor the exchanger depends on both the electrical properties of theprotein, and the relative affinity of other charged substances in thesolvent. Hence, bound proteins can be eluted by changing the pH, thusaltering the charge of the protein, or by adding competing materials, ofwhich salts are but one example. Because different substances havedifferent electrical properties, the conditions for release vary witheach bound molecular species. In general, to get good separation, themethods of choice are either continuous ionic strength gradient elutionor stepwise elution. For an anion exchanger, either pH is decreased andionic strength is increased or ionic strength alone is increased. For acation exchanger, both pH and ionic strength can be increased. Theactual choice of the elution procedure is usually a result of trial anderror and of considerations of stability of the VLPs being purified.

It will be appreciated by a skilled practitioner of this art, that thetype of anion-exchanger, and the buffers, and salts used to bind andelute the VLP will also be a function of the type of VLP sought to bepurified.

Cation Exchange Chromatographic Material

In cation exchange chromatography, a negative functional group is boundto the insoluble support medium. Accordingly, cation exchangechromatographic media bind positive counter ions when the incubationperiod is a sufficient time period to allow for the positively chargedgroups to bind to and come to equilibrium with the negatively chargedcation exchanger medium. Neutral molecules and anions do not bind to thecation exchange medium. Following the electrostatic binding of speciespossessing a net positive charge, the cationic medium is washed,removing non-binding molecules from the medium. Bound ions are theneluted either by washing the medium with increasing concentrations ofpositive ions or by altering the pH of the medium. The disclosedinvention contemplates using a variety of cation exchange media such asany sulfo-, phosphor carboxy-, or carboxy-methyl-based cation exchangeresins bound to numerous support medium well known in the art.

In one embodiment of the invention, ion exchange chromatography may beused in binding mode or flow-through mode discussed below.

Affinity Chromatographic Material

In some embodiments, the chromatographic material comprises an affinitychromatographic material. The affinity chromatographic material maycomprise antibodies, dye resins, and metals. The dry resin may becibachrom blue or polymixin.

Affinity chromatography is a technique that provides for ligand specificpurification of a target compound. As such, the technique exploits thestructural and functional characteristic properties of macromolecules bybinding the molecules based on these specific characteristics undercertain conditions.

A variety of different affinity column matrices are contemplated for usewith the disclosed invention. For example, antibodies directed againstVLPs may be used to generate affinity column media that in turn can beused to purify VLPs. In addition, the affinity chromatographic materialmay comprise dry resins, and metals. The dry resin may be cibachrom blueor polymixin.

One embodiment of the disclosed invention contemplates the use ofheparin as the adsorbent group. Affinity chromatography media containingheparin are commercially available from a variety of sources. Forexample, PerSeptive Biosystems, Inc. (Framingham, Mass.) markets aheparin-based medium (POROS 20HE™). When POROS 20HE™ is used as theaffinity chromatography medium, the VLPs containing solution is appliedto the affinity medium and subsequently eluted with an appropriate saltconcentration.

The chromatographic materials discussed above can be run as individualsteps, or sequentially, or in tandem. The sequence of the multistepchromatographic process of the present invention may be designed toproduce VLPs meeting preset specifications. For example, thepurification method of the present invention may be used to purify VLPsto greater than about 70%, 80, 90%, 95% or 99% purity.

In one embodiment, the sequence of the multistep chromatographic processis designed to control the resulting composition of VLPs. In anotherembodiment, the sequence of the multistep chromatographic process isdesigned to reduce contaminant levels to levels considered acceptable byregulatory agencies for pharmaceutical grade drug substance. Forinstance, the contaminant level of the host cell DNA content may bereduced to less than 1%. The contaminant level of the host cell proteincontent may be reduced to less than 5%. The sequence of the multistepchromatographic process is designed to make VLPs consistent with cGMPregulations and suitable for pharmaceutical testing in humans.

The present invention further provides a method of contacting a solutioncontaining VLPs with a chromatographic material. In this regard, culturemedia or a cell lysate containing VLPs may be contacted with thechromatographic material wherein the culture media or cell lysate isfiltered or purified by precipitation prior to contact with thechromatographic material. The solution or cell lysate may be centrifugedwithout a sucrose gradient. In one embodiment, a clarified solutioncontaining VLPs is contacted with the chromatographic material.Alternatively, a VLP containing solution from one chromatographic stepis contacted with another chromatographic material.

Certain embodiments of the disclosed invention contemplate the use of ahydroxyapatite medium in conjunction with a hydrophobic interactionchromatography medium to purify VLPs particles from the cellular milieureleased during the lysis process. In one embodiment, the cell lysate isloaded on a hydroxyapatite column (Bio-Rad, CHT). Such columns areavailable commercially in a variety of sizes. Following purificationover the hydroxyapatite column, the VLPs-containing material is passedover a hydrophobic interaction (HIC) column. The column is then washedand eluted. The purified sample of VLPs can be analyzed, for example, bysilver-stained SDS-PAGE or size exclusion chromatography (SEC) forpurity.

In one embodiment of the invention, a hydroxyapatite medium is used inconjunction with a hydrophobic interaction chromatographic medium andfurther in conjunction with a Anion exchange chromatographic medium inorder to purify VLPs particles for pharmacological use. The presentinventors have found that this combination is particularly suitable forpurifying Norovirus Genotype I Norwalk viruses on a commerciallyscalable level. In addition, the present inventors have found thatcation exchange chromatography followed by methyl HIC is particularlysuitable for purifying Houston viruses.

Before contacting the chromatographic material with the VLP preparationin each step, it may be necessary to adjust parameters such as pH, ionicstrength, and temperature and in some instances the addition ofsubstances of different kinds. Adjustment of these parameters is withinthe knowledge of one skilled in the art and may be accomplished in theVLP containing solution or chromatographic medium. For example, the pHof the VLP containing solution may be adjusted with a buffer. The pH maybe adjusted to either acidic values (e.g. pH less than 7) or basicvalues (e.g. pH greater than 7). In some embodiments, it may bedesirable to adjust the pH of the solution or chromatographic materialto a neutral value (e.g. pH equal to 7). The buffer used to adjust thepH value may comprise phosphate, carboxylate, sulfate, acetate, citrate,tris or his tris and the buffer concentration may be in the range ofabout 10 to 1000 mM.

In another embodiment, the method of the present invention involvesadjustment of the ionic strength of the VLP containing solution. Theionic strength may be adjusted by the addition of a salt comprisingcations and anions from the Hofineister series. The salt may be aphosphate salt, such as sodium phosphate, calcium phosphate, andpotassium phosphate. In some embodiments, the phosphate salt is sodiumphosphate. The concentration of the salt may be in the range of about 10to 500 mM. In one embodiment, the sodium phosphate concentration isabout 100 mM.

Alternatively, an optional step may be performed on a chromatographicmaterial by washing it with a solution (e.g., a buffer for adjusting pH,ionic strength, etc., or for the introduction of a detergent) to bringthe necessary characteristics for purification of the VLP preparation.For example, the pH of the chromatographic material may be adjustedprior to VLP application by equilibration with a buffer. The pH may beadjusted to less than 7 or greater than or equal to 7 with a buffercomprising phosphate, carboxylate, or sulfate.

In another embodiment, the ionic strength of the chromatographicmaterial may be adjusted by the addition of salt. The salt comprisescations and anions from the Hofineister series such as ammonium sulfate.The concentration of ammonium sulfate may be greater than about 1, 2, or2.4 molar.

In another embodiment, the ionic strength of the chromatographicmaterial may be adjusted by the addition of a phosphate salt. In someembodiments, the phosphate salt is sodium phosphate. The sodiumphosphate concentration may be in the range of about 10 to 500 mM. Inone embodiment, the sodium phosphate concentration is about 100 mM. Inyet another embodiment, the organic solvent concentration of the VLPcontaining solution can be adjusted during reversed phase processes.

Adjustment of the parameters of the VLP containing solution orchromatographic material can cause retention or pass-through of VLPs andcontaminants. In one embodiment, the VLP containing solution orchromatographic material may be selected to cause retention of VLPs withcontaminating materials passing through the chromatographic material. Inanother embodiment, the VLP containing solution or chromatographicmaterial may be selected to cause retention of contaminating materialswith VLPs passing through the chromatographic material. Such featurescan be utilized to operate the chromatographic steps in either“flow-through mode” or “binding mode” or a mixture thereof. The term“flow-through mode” refers to a VLP preparation separation technique inwhich at least one VLP contained in the preparation is intended to flowthrough a chromatographic resin or support, while at least one potentialcontaminant or impurity binds to the chromatographic resin or support.Flow-through mode may be used, for instance, in hydroxyapatitechromatography and ion exchange chromatography.

“Binding mode” refers to a VLP preparation separation technique in whichat least one VLP contained in the preparation binds to a chromatographicresin or support, while at least one contaminant or impurity flowsthrough. Binding mode may be used, for instance, in hydroxyapatitechromatography and ion exchange chromatography.

In certain embodiments, the present invention provides methods forremoving contaminating materials from VLP preparations usinghydroxyapatite chromatography or hydrophobic interaction chromatographyin binding mode, flow-through mode, or a combination thereof. Suchpractice has application to the large scale purification of VLPpreparations.

In binding mode hydroxyapatite chromatography, the method uses ahydroxyapatite support charged with phosphate at neutral pH and lowionic strength to bind VLPs. The column is then washed with a phosphatebuffer to remove loosely bound impurities. Next, the VLPs areselectively eluted using a high ionic strength phosphate buffercontaining 100 to 200 mM phosphate. Lastly, the resin is optionallyregenerated using a sodium hydroxide and potassium phosphate solution.

In flow-through mode hydroxyapatite chromatography, a VLP preparation isbuffer-exchanged into a load buffer at an appropriate pH. The VLPpreparation is then allowed to flow through a hydroxyapatite column,while impurities bind to the column. The column is optionallysubsequently washed and cleaned to allow additional VLPs to flow throughthe column and be purified. Lastly, the column may optionally bestripped and then regenerated using buffer such as a sodium hydroxideand potassium phosphate solution.

In combination binding/flow-through mode hydroxyapatite chromatography,the hydroxyapatite media is equilibrated and washed with a solution,thereby bringing the necessary characteristics for purification of theVLP preparation.

Prior to equilibration and chromatography, the hydroxyapatitechromatography medium may be pre-equilibrated in a chosen solution, e.g.a salt and/or buffer solution. Pre-equilibration serves the function ofdisplacing a solution used for regenerating and/or storing thechromatography medium. One of skill in the art will realize that thecomposition of the pre-equilibration solution depends on the compositionof the storage solution and the solution to be used for the subsequentchromatography. Thus, appropriate pre-equilibration solutions mayinclude the same buffer or salt used for performing the chromatography,optionally, at a higher concentration than is used to performchromatography.

Before the sample is applied to the column, the hydroxyapatitechromatography medium can be equilibrated in the buffer or salt thatwill be used to chromatograph the VLP. Chromatography (and loading ofthe protein to be purified) can occur in a variety of buffers or saltsincluding sodium, potassium, ammonium, magnesium, calcium, chloride,fluoride, acetate, phosphate, and/or citrate salts and/or Tris buffer.Such buffers or salts can have a pH in a range from about 2 to about 10.In some embodiments, equilibration may take place in a solutioncomprising a Tris or a sodium phosphate buffer. Optionally, the sodiumphosphate buffer is at a concentration between about 0.5 millimolar andabout 50 millimolar, more preferably at a concentration between about 15millimolar and 35 millimolar. Preferably, equilibration takes place at apH of at least about 5.5. Equilibration may take place at pHs betweenabout 6.0 and about 8.6, preferably at pHs between about 6.5 and 7.5.Most preferably, the solution comprises a sodium phosphate buffer at aconcentration of about 25 millimolar and at a pH of about 6.8.

The contacting of a VLP preparation to the hydroxyapatite resin ineither binding mode, flow-through mode, or combinations thereof may beperformed in a packed bed column, a fluidized/expanded bed columncontaining the solid phase matrix, and/or in a simple batch operationwhere the solid phase matrix is mixed with the solution for a certaintime.

After contacting the hydroxyapatite resin with the VLP preparation thereis optionally performed a washing procedure. However, in some cases, thewashing procedure may be omitted, saving a process-step as well aswashing solution. The washing buffers employed will depend on the natureof the hydroxyapatite resin, the mode of hydroxyapatite chromatographybeing employed, and therefore can be determined by one of ordinary skillin the art. In flow-through mode and combination binding/flow-throughmode, the purified VLP flow-through obtained after an optional wash ofthe column may be pooled with other purified VLP fractions.

In binding mode, the VLP may be eluted from the column after an optionalwashing procedure. For elution of the VLP from the column, thisinvention uses a high ionic strength phosphate buffer. For example, theelution buffer may contain 1 to 300 mM sodium phosphate, in anotherembodiment it may contain 50 to 250 mM sodium phosphate, in anotherembodiment it may contain 100 to 200 mM sodium phosphate, in anotherembodiment may contain 150 mM sodium phosphate. The pH of the elutionbuffer may range from 6.4 to 7.6. In one embodiment, the pH may be from6.5 to 7.3, in another embodiment the pH may be 7.2, and in anotherembodiment the pH may be 6.8. The elution buffer may be altered forelution of the VLP from the column in a continuous or stepwise gradient.Buffer components may be adjusted according to the knowledge of theperson of ordinary skill in the art.

In both binding, flow-through mode, and combinations thereof, a solidphase matrix may optionally be cleaned, i.e. stripped and regenerated,after elution or flow through of the VLP. This procedure is typicallyperformed regularly to minimize the building up of impurities on thesurface of the solid phase and/or to sterilize the matrix to avoidcontamination of the product with microorganisms.

In certain embodiments, the hydroxyapatite chromatography step isconducted first, the hydrophobic interaction chromatography step isconducted second, and either anion exchange or size exclusionchromatography step is conducted third.

VLPs are generally recovered from an hydroxyapatite step in thefractions of binding, flow-through or a mixture thereof. The saltconcentration and pH of such fractions can then be adjusted forpurification over a hydrophobic interaction column or for purificationover any other suitable affinity column, as described in Example 1. Inaccordance with this method, ammonium sulfate is added to a finalconcentration of 8% w/v to pooled VLPs and the sample is stirred untilall of the ammonium sulfate is dissolved. A column containing 250 mL ofHIC resin is equilibrated with five CV of 100 mM sodium phosphate, 2.4 Mammonium sulfate pH 6.8 and the VLP suspension loaded. The column iswashed with approximately three CV of buffer C until a stable baselineis observed and then washed with 10 CV of 70% 100 mM phosphate, pH 6.8.The VLPs can be eluted from the column with 150 mM phosphate.

It should be noted that the order of the chromatographic media is notconsidered to be important to the ultimate purification of the VLPsparticles. Also, a size exclusion column may optionally be used tofurther purify the sample. The yields obtained using such combinationsare predictable based on the yields obtained using the individualcolumn-purification steps.

VLPs eluted from HIC column can be subjected to additional sizeexclusion purification steps as required. In one embodiment of theinvention, eluate purified by combination hydroxyapatite chromatographyand HIC chromatography was further purified by a DEAE anion exchangechromatography.

Any or all chromatographic steps of the invention can be carried out byany mechanical means. Chromatography may be carried out in a column. Thecolumn may be run with or without pressure and from top to bottom orbottom to top. The direction of the flow of fluid in the column may bereversed during the chromatography process. Chromatography may also becarried out using a batch process in which the solid support isseparated from the liquid used to load, wash, and elute the sample byany suitable means, including gravity, centrifugation, or filtration.Chromatography may also be carried out by contacting the sample with afilter that adsorbs or retains some molecules in the sample morestrongly than others.

Finally, the eluate from the Anion exchange column may be filtratedthrough a diafilter or a tangential flow filter to concentrate thefiltrate.

Additional Optional Steps

Although it has been discovered that hydroxyapatite and hydrophobicinteraction chromatography can be used together to separate VLP, asmentioned above, the purification method of the invention can be used incombination with other protein purification techniques. In oneembodiment of the invention, one or more steps preceding thehydroxyapatite step may be desirable to reduce the load challenge of thecontaminants or impurities. In another embodiment of the invention, oneor more purification steps following the hydrophobic interaction stepmay be desirable to remove additional contaminants or impurities.

The hydroxyapatite purification procedure described may optionally becombined with other purification steps, including but not limited to,Protein A chromatography, affinity chromatography, hydrophobicinteraction chromatography, immobilized metal affinity chromatography,size exclusion chromatography, diafiltration, ultrafiltration, viralremoval filtration, and/or ion exchange chromatography.

Further purification methods may include filtration, precipitation,evaporation, distillation, drying, gas absorption, solvent extraction,press extraction, adsorption, crystallization, and centrifugation. Otherpurification methods may include further chromatography according tothis invention utilizing batch or column chromatography. In addition,further purification can include combinations of any of the forgoing,such as for example, combinations of different methods ofchromatography, combinations of chromatography with filtration,combinations of chromatography with precipitation, or combinations ofmembrane treatment with drying.

Elution of VLPs can be monitored by techniques known in the artincluding optical density, transmission electron microscopy, or lightscattering. Additionally, the biological properties of the VLPs prior toand after purification can be determined using well established assays.More specifically, the VLP purity and identity may be measured using avariety of analytical methods including, reduced and non-reducing sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sizeexclusion chromatography, HPLC (high performance liquid chromatography),capillary electrophoresis, MALDI (Matrix Assisted Laser DesorptionIonization) mass spectrometry, ELISA (Enzyme Linked ImmunosorbentAssay), or circular dichroism.

The following examples are included to demonstrate preferred embodimentsof the invention. A skilled practitioner of the art would, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments and obtain a similar result without departing fromthe spirit and scope of the invention.

Example 1 Purification of Norovirus Genogroup I Viruses GeneralDescription of Method

The purification process consists of 3 chromatography steps. The stepsuse orthogonal mechanisms resulting in a scalable process that produceshighly purified VLPs. The first step of the purification is a capturestep that uses Bio-Rad hydroxyapatite (CHT) resin. The CHT stepconcentrates Norwalk VLPs, eliminates media components, and exchangesthe product to a phosphate buffer. Following the addition of ammoniumsulfate, methyl hydrophobic interaction chromatography (HIC) providesthe majority of the purification as shown in FIG. 1. The thirdchromatographic step is a DEAE ion exchange chromatography operated inin-flow mode. Under the conditions used, Nowalk VLPs do not bind to thecolumn and residual contaminants (endotoxin, nucleic acid, and proteins)are retained. The final step in the purification process is an ultrafiltration where phosphate buffer is replaced by water for injection.Bulk drug substance (Norwalk VLPs) is stored as a 0.5 to 1.5 mg/mLsuspension at 2-8° C.

A number of tests on the chromatographically-purified VLPs can beconducted to determine whether there is any difference between thechromatographically-purified VLPs and ultracentrifuge-purified VLPs.These methods include transmission electron microscopy andphysical/chemical characterization, including melting points by circulardichroism spectra, dynamic light scattering, size exclusionchromatography and high performance liquid chromatography. Thechromatography purification procedures are outlined below. Thepurification process can be normalized for any scale. Linear flow rateslisted are independent of column diameter.

Purification of VLPs

A 15 L fermentation produces 10 L of conditioned media. The purificationprocess described below is designed to handle 5 L. To purify the entire10 L, 2 campaigns using 5 L each are performed over a two week period.

HA Column Chromatography:

Five liters of culture supernatant is loaded onto a column packed with500 mL of hydroxyapatite (Bio-Rad, CHT) resin, equilibrated by passing10 column volumes of 5 mM sodium phosphate buffer (buffer A) with a flowrate of 80 mL/min. The culture supernatant, is loaded onto the columnand washed with two column volumes (CV) of buffer A. 150 mM phosphate(buffer B) is used to elute the Norwalk VLPs from the CHT column. TheNorwalk VLPs elute as a single peak off in buffer B. Fractions (1 CVeach) are collected during the chromatographic run and analyzed bySDS-PAGE. The fractions containing VLPs are pooled for the next step inthe purification. SDS-PAGE with Coomassie Stain of Hydroxyapatitechromatography fractions are shown in FIG. 2. The majority of theNorwalk VLPs elute in the 4 eluate column volume fractions with the 100%buffer B. These pooled fractions were used for the hydrophobicinteraction chromatography (HIC) purification step 2

HIC Column Chromatography:

To the pooled VLP fractions from the CHT chromatography step, solidammonium sulfate is added to a final concentration of 8% w/v and thesample stirred until all of the ammonium sulfate is dissolved. Additionof ammonium sulfate facilitates absorption of the protein onto BioRadMethyl HIC resin. A column containing 250 mL of HIC resin isequilibrated with five CV of 100 mM sodium phosphate, 2.4 M ammoniumsulfate pH 6.8 (buffer C) and the VLP suspension loaded. The column iswashed with approximately three CV of buffer C until a stable baselineis observed and then washed with 10 CV of 70% 100 mM phosphate, pH 6.8(buffer D). VLPs from the HIC Column elute in three to four CV of 100%buffer B. During elution 250 mL (1 CV) fractions are collected andanalyzed by SDS-PAGE. Fractions containing VLPs are pooled and used inthe DEAE chromatographic step. SDS-PAGE/Coomassie stain of HIC columnfractions are shown in FIG. 3.

DEAE Column Chromatography:

Pooled fractions, containing the partially purified VLPs from the methylHIC column, are loaded directly onto a column packed with 270 mL DEAESephadex resin. Phosphate buffer pH 6.5 is pumped at a flow rate of 40mL/min causing the VLPs to elute in the void volume (flow through).Contaminating proteins interact with the resin and elute later in thechromatography. Fractions (¼ CV each) are collected upon sample loadingand as soon as UV detector signal rises above baseline. Fractions areanalyzed by SDS-PAGE and fractions containing VLPs pooled for bufferexchange by Diafiltration. SDS-PAGE/Coomassie stain of DEAE columnfractions are shown in FIG. 4.

Diafiltration:

In the final purification step Norwalk VLPs, which elute from the DEAEcolumn are diafiltered and concentrated. This procedure involves placingthe VLPs in a sanitized stirred cell diafiltration apparatus. The volumeof liquid is reduced by 50% and water for injection is added back to theoriginal volume. This process was repeated a total of 10 times. Theretentate contains the diafiltered Norwalk VLPs. This material then goesthrough a final sterile filtration process. Aliquots are taken for QCtesting and release. SDS-PAGE/Coomassie stain from diafiltrationfractions are shown in FIG. 5.

VLP Content:

Throughout purification, coomassie stained SDS-PAGE gels are used toidentify fractions containing VLPs. During purification, a fraction isassumed to contain VLPs if a band is observed that migrates with asimilar molecular weight as a VLP reference material run on the samegel. Only those fractions where the VLP band has an intensity equivalentto or greater than the intensity of VLP standard are pooled.

Protein Concentration:

In-process protein concentration (ultra filtration step) is determinedby chromogenic assay based on bicinchonic acid reaction with protein.Test reagents are obtained from Pierce.

The specifications of the Norwalk virus VLPs purifiedchromatographically by the method described above are shown in Table 1.The chromatographically purified Norwalk virus VLPs were also comparedto the VLPs purified by the ultracentrifugation method usingtransmission electron microscopy (TEM) and CD spectra. The results areshown in FIGS. 6-13.

TABLE 1 Test Specification Result Identify Confirmed for a. Molecularweight Complies by SDS-PAGE protein band between 49 and 62 KDa b. 49 to62 kDa protein Complies detected by Western blot Protein 0.5 mg/mL to1.5 mg/mL 1.40 mg/mL Concentration Purity Greater than 90%  >99% sizeexclusion chromatography Host Cell DNA <100 pg/mL <100 pg/mL BaculovirusDNA <100 pg/mL  >31 & <62 pg/mL Host Cell Protein Not more than 5% <0.3%Endotoxin Less than 640 EU/mL >320 & <640 EU/mL pH ≦7.0 5.4

Example 2 Purification of Norovirus Genogroup II Viruses GeneralDescription of Method

The Houston purification process also utilizes orthogonal mechanismsresulting in a scalable process that produces highly purified VLPs. Inconstrast to the Norwalk purification process that utilizes HA, HIC andAnion Exchange, we find that a 2 column process works well for genogroup11.4 Houston virus VLPs.

To purify Houston VLPs, conditioned media containing VLPs is clarifiedby either filtration or centrifugation and loaded on to a cationexchange SP FF resin equilibrated with 20 mM citrate phosphate buffer,pH 4.0. Following a wash with 20% elution buffer (20 mM citratephosphate, 1M sodium chloride), the VLPs are eluted using step gradientand 100% elution buffer.

The fractions from the cation exchange column which contain Houston VLPsare pooled and the ionic strength adjusted through the addition of 15%(w/v) ammonium sulfate. This pooled material is then loaded onto acolumn containing methyl HIC resin with phosphate buffer at pH 6.8containing 2.4 M ammonium sulfate (buffer A). After loading, a 3 stepgradient elution is used to elute the VLPs from the HIC resin. In thefirst step, 40% elution buffer (100 mM sodium phosphate) is used toelute contaminants. Next, the gradient is stepped up to 70% elutionbuffer which causes the VLPs to elute and finally the gradient isadjusted to 100% elution buffer to ensure that elution is complete andto elute any remaining contaminants. The elution peak containing VLPsfrom methyl HIC chromatography in 70% elution buffer is then dialyzedinto 20 mM citrate phosphate buffer and 150 mM sodium chloride to pH6.0.

Based on small-scale production runs, the process results in 5 to 15 mgof purified VLPs from 200 and 500 mL spinner flasks and results in a VLPpurity of greater than 90% by SDS-PAGE.

Table 2 summarizes this purification process for the genogroup IINorovirus, Houston virus. FIGS. 14 and 15 provide the results from thecation exchange step, whereas FIGS. 16 and 17 provide the results fromthe methyl HIC chromatography step. FIGS. 18 and 19 provide an SDS-PAGEand HPLC-SEC of the purified Houston virus protein.

TABLE 2 Culture MOI = 1 Conditions 3 × 10⁶ cells/mL Add NaCl to 150 mMon Day 7 Harvest the supernatant Purification 1^(st) step: Cationexchange Equilibration Buffer: 20 mM Protocol SP FF resin citratephosphate ph 4.0 Elution Buffer: 20 mM citrate phosphate, 1M sodiumchloride pH 4.0 Step Gradient: 1^(st) step - wash with 20% elutionbuffer; 2^(nd) step - elution with 100% elution buffer 2^(nd) step:Methyl HIC Equilibration Buffer: 100 mM resin-start material sodiumphosphate, 2.4M contains 15% ammonium sulfate pH 6.8 ammonium sulfateElution Buffer: 100mM sodium phosphate pH 6.8 Sample loaded with theaddition of ammonium sulfate to 15% (w/v). Step Gradient: 1^(st) step -wash with 40% elution buffer; 2^(nd) step - elution with 70% elutionbuffer; 3^(rd) step - wash with 100% elution buffer. 3^(rd) step:Dialysis Elution peak obtained from to buffer methyl HIC chromatographywith 70% elution buffer dialyzed into 20 mM citrate phosphate buffer,150 mM sodium chloride pH 6.0. Final 20-30 mg purified Yields protein/L

In addition to the process outlined in Table 2, we have been able toselectively precipitate Houston VLPs by decreasing the ionic strength ofthe conditioned media through the addition of deionized water. Thisoffers a significant advantage because of the quick reduction in volumeafforded by precipitation processes. Once precipitated, the VLPs can beseparated using either centrifugation or filtration. Precipitated VLPsare then resuspended in an appropriate buffer and purified further usingthe chromatographic methods described above. We envision developing ascalable purification method using at least two and maybe all three ofthese chromatography techniques. The objective of this purificationdevelopment would be to obtain a purification scheme that would providefunctional VLPs that are identical in quality to the VLPs normallyproduced via ultracentrifugation and could be produced at large scalefor commercial manufacture of the Houston VLPs. The preliminary datapresented above demonstrate that chromatographic methods will be usefulfor purification of Houston virus VLPs. The VLPs were shown to bind toseveral resins and could also be eluted with the appropriate buffers.The next step in development will be to determine the combination andorder in which these chromatographies will be used to produce a purerproduct. Also, additional experiments will be done to determine themodifications required to these chromatography techniques to obtain themaximum yield of VLPs. The finalized method will then be used in thelarge scale production and purification of Houston virus VLPs.

Example 3 Purification of G.II VLPs by Ammonium Sulfate Precipitation

A partially purified preparation of Houston VLPs was divided into 1 mLaliquots. Ammonium sulfate was added to each aliquot to achieve a finalconcentration of 10% to 35% (w/v). The samples were placed in a rotatorand mixed end over end at 4° C. overnight. The samples were visuallyinspected for signs of precipitation. An aliquot of 20 μl, was removedand labeled “Amm suspension”. The ammonium sulfate suspension wascentrifuged at 14,000×g for 10 min, room temperature. The resultingsupernatant was pulled and labeled “Amm Supt”. The precipitated pelletwas dissolved in a citrate buffer, pH 7.0, placed in a rotator and mixedend over end at 4° C. for 2 h. The samples were centrifuged at 14,000×gfor 10 min. The resulting supernatant was pulled and labeled “Citratesupt”. FIG. 20 depicts a silver stained SDS-PAGE gel of samples taken atdifferent points through the purification process. The two bands in the“Citrate supt” lane reflect the purified Houston VLPs.

The ammonium sulfate precipitation step significantly improved thepurity of the VLPs as shown in FIG. 21. The percentage of host cellprotein (HCP) to VLP protein was reduced by approximately half as aresult of the ammonium sulfate precipitation.

Example 4 Purification of Norovirus Genogroup II VLPs by PrecipitationFollowed by Quaternary Amine (Q) Chromatography Houston VLP PurificationProcess

A 1 liter suspension culture of SF9 cells was grown up to a density of1.7×10⁷ cells/mL and infected at a MOI (multiplicity of infection) of0.5 pfu/cell with recombinant Baculovirus stock encoding for the HoustonVP1 sequence. The infection was allowed to proceed to a viability ofless than 20% (FIG. 22, lane 2). The Houston VLPs were harvested,purified, and concentrated in the following manner. The pH of theculture was lowered to 5.5 using HCl (lane 3) to precipitate the VLPs.Then the culture at pH 5.5 was centrifuged at 1,000×g for 5 minutes atroom temperature. The supernatant (lane 4) was decanted and discarded.Next, the remaining pellet was resuspended in 20 mM Tris, 50 mM NaCl, 10mM EDTA, at pH 8 (lane 5). The resuspended pellet was then centrifugedat 1,000×g for 5 minutes. The supernatant was decanted and labeled“Houston VLP extract” (lane 6). The precipitation process resulted in asignificant improvement in purity as illustrated by the single band inlane 6 as compared to the multiple bands observed in the startingmaterial in lane 2 (FIG. 22).

Following harvest, the Houston VLP extract was diluted 1:2 with waterand loaded onto a Q100 membrane. The mobile phase buffer was 20 mM TrispH 6.5 and the elution buffer was 20 mM Tris, 1M NaCl pH 6.5. Afterloading, the column was washed with 20% elution buffer (FIG. 22, lane 7)followed by 50% elution buffer (lane 8). The silver stained SDS-PAGE geldepicted in FIG. 22 shows that the Houston VLPs are concentrated by thiscapture chromatography step (compare lane 8 to lane 2).

Laurens VLP Purification Process

The VLP harvest process outlined above for the Houston VLPs was appliedto a SF9 culture infected with recombinant Baculovirus stock encodingfor the VP1 sequence from the GII.4 Laurens virus, resulting in aLaurens VLP extract (FIG. 23, lane 11). The resuspended pellet wasdiluted 1:2 with water and loaded onto a Q100 membrane. The mobile phasebuffer was 20 mM Tris pH 6.8 and the elution buffer was 20 mM Tris, 1MNaCl pH 6.8. Lanes 2 through 10 of FIG. 23 illustrate the fractionscollected over a step gradient elution from the Q100 membrane. The VLPseluted in the 40% elution buffer fractions (lanes 5-8). As observed withHouston, the Laurens VLPs could be purified and concentrated using pHadjustment and capture chromatography.

Example 5 pH-Dependent Changes in Norovirus GI and GII VLP Structure

To further optimize VLP purification procedures, the stability of VLPsfrom Norovirus GI and GII strains were exposed to pH ranges from aroundpH 2 to around pH 10.

Intact VLPs are of approximately 10 MDa mass. Intact VLPs exposed tocertain pH conditions and analyzed by size exclusion high performanceliquid chromatography (SE-HPLC) show a transition to a stableintermediate fragment with a mass of around 220 kDa relative to globularprotein size standards. Fully denaturing conditions cause furtherdisassembly of the intermediate fragment to the monomer of around 60kDa. To further explore the pH range in which the Norovirus VLPs wouldremain intact, the pH of solutions containing either Norovirus GI VLPsor Norovirus GII VLPs was adjusted incrementally from about 2 to about10 prior to analysis with SE-HPLC. Additionally, the SE-HPLC columnbuffer was adjusted to the same pH for analysis.

Norovirus GI VLPs exposed to pH conditions ranging from pH 2 (FIG. 24A)to pH 8 (FIG. 24B) exhibit a single absorbance peak at about 17 minrepresenting intact, monodisperse VLPs. VLPs exposed to pH 8.5 (FIG.24C) produce an absorbance peak at about 33 min, which represents thelower order stable intermediate VLP fragments. These results show thatNorovirus GI VLPs remain intact from pH 2 to 8, and disassemble to thestable intermediate fragment between pH 8 and 8.5.

Norovirus GII VLPs exposed to pH conditions ranging from pH 2 (FIG. 25A)to pH 9.5 (FIG. 25B) produce single absorbance peaks at about 17 min,while VLPs exposed to pH 10 (FIG. 25C) exhibit single absorbance peaksaround 34 min. Thus, these results show that Norovirus GII VLPs remainintact over the range of pH 2 to 9.5, and disassemble to the stableintermediate fragment between pH 9.5 and 10.

The findings of these experiments allow one to select conditions thatresult in intact VLPs or fragments thereof by adjusting the pH of thesolution containing the VLPs. Such conditions may facilitate thepurification process.

Example 6 Precipitation of VLP Proteins Using Polyethylene Glycol (PEG)

In a manner similar to that observed for the precipitation of VLPs bythe addition of salt (Example 3), similar results may be produced byusing other additives, such as polyethylene glycol, to cause theselective precipitation and solubilization of VLPs.

To determine the optimal concentration of polyethylene glycol (PEG) massand concentration, PEG with molecular masses ranging from 200 to 20,000is added to solutions containing VLPs in amounts that result in finalPEG concentrations of 0 to 50% PEG in 5% increments. Following each 5%PEG addition, the VLP containing solutions are centrifuged at 10,000 g,and samples of the supernatants are obtained. The supernatant samplesare subjected to SDS-PAGE, ELISA, and/or HPLC analyses to evaluate thepurity and total concentration of the VLPs.

The VLPs will initially be found in the supernatant. As the PEGconcentration increases, the VLPs and contaminating proteins willprecipitate to varying degrees. When pellets are observed, the pelletsare collected by decanting the supernatant. The pellet is resuspended inbuffer and the purity of the VLPs analyzed by SDS-PAGE. The total VLPcontent in the resuspended pellets is evaluated by ELISA or HPLC. Atable or graph of the relative purity of the supernate and pellet versusPEG mass and concentration is prepared. The optimal PEG mass and PEGconcentration combination is selected based on the conditions resultingin the highest purity with acceptable yield or product recovery.

1-118. (canceled)
 119. A method of purifying Calicivirus virus-likeparticles (VLPs) using a multistep chromatographic process, said methodcomprising (a) contacting a cell lysate or culture supernatantcontaining said VLPs with an ion exchange chromatographic material,wherein said VLPs bind to said ion exchange chromatographic material;(b) contacting the VLPs eluted from said ion exchange chromatographicmaterial with a hydrophobic interaction chromatographic material,wherein said VLPs bind to said hydrophobic interaction chromatographicmaterial; and (c) eluting said VLPs from said hydrophobic interactionchromatographic material, wherein said VLPs are purified to greater thanabout 70%, wherein the chromatographic materials used in the method donot comprise hydroxyapatite.
 120. The method of claim 119, wherein theion exchange chromatographic material comprises a cation-exchanger. 121.The method of claim 119, wherein the hydrophobic interactionchromatographic material is a methyl HIC resin.
 122. The method of claim119, wherein the ionic strength of the VLP-containing solution in step(b) is adjusted to cause the retention of VLPs with contaminatingmaterials passing through the hydrophobic interaction chromatographicmaterial.
 123. The method of claim 122, wherein the ionic strength isadjusted by the addition of ammonium sulfate.
 124. The method of claim119, wherein the eluate from step (c) is ultrafiltered and/ordiafiltered.
 125. The method of claim 119, further comprising (d)contacting the eluate from step (c) with a third chromatographicmaterial, wherein contaminating materials are retained and said VLPspass through the third chromatographic material; and (e) collecting saidsolution containing said VLPs.
 126. The method of claim 125, wherein thethird chromatographic material comprises an anion-exchanger.
 127. Themethod of claim 125, wherein the solution containing said VLPs from step(e) is ultrafiltered and/or diafiltered.
 128. The method of claim 125,wherein said VLPs are purified to greater than about 90%.
 129. Themethod of claim 119, further comprising contacting the VLPs eluted fromsaid ion exchange chromatographic material with an adsorption materialprior to contact with the hydrophobic interaction chromatographicmaterial, wherein contaminating materials are retained and said VLPspass through the adsorption material.
 130. The method of claim 119,wherein the Calicivirus VLPs are Norovirus VLPs.
 131. The method ofclaim 130, wherein the Norovirus VLPs are Norovirus genogroup I orgenogroup II VLPs.
 132. The method of claim 131, wherein the Norovirusgenogroup I VLPs are Norovirus genotype I.1 VLPs.
 133. The method ofclaim 119, wherein the cell lysate or the culture supernatant isproduced using recombinant methodologies.
 134. The method of claim 119,wherein the contaminant level of host cell DNA content from the celllysate or culture supernatent is less than 1%.
 135. The method of claim119, wherein the contaminant level of host cell protein content from thecell lysate or culture supernatent is less than 5%.
 136. The method ofclaim 1, wherein the cell lysate or the culture supernatant is filtered,centrifuged, or clarified prior to contact with the ion exchangechromatographic material.
 137. The method of claim 136, wherein the celllysate or the culture supernatant is clarified by precipitation or bysize exclusion chromatography.
 138. The method of claim 119, wherein theVLPs are eluted after contacting said ion exchange chromatographicmaterial in buffer at a pH of 4.0-9.0
 139. The method of claim 138,wherein the buffer has an effective capacity within the range of pH4.0-9.
 140. A pharmaceutical agent comprising VLPs prepared by a methodaccording to claim
 119. 141. The pharmaceutical agent of claim 140,wherein the pharmaceutical agent comprises a vaccine.
 142. A method ofpurifying Calicivirus virus-like particles (VLPs) comprising contactinga solution containing said VLPs with two or more chromatographicmaterials, wherein the chromatographic materials do not comprisehydroxyapatite, and wherein at least one chromatographic materialretains said VLPs.
 143. The method of claim 142, wherein the two or morechromatographic materials are selected from ion-exchange, affinity,hydrophobic interaction, mixed mode, reverse phase, size exclusion oradsorption material.
 144. The method of claim 143, wherein the two ormore chromatographic materials are ion-exchange and hydrophobicinteraction.
 145. The method of claim 144, wherein the ion-exchangematerial comprises a cation-exchanger.
 146. The method of claim 145,wherein the two or more chromatographic materials further comprise ananion-exchanger.
 147. The method of claim 144, wherein the ion exchangematerial comprises an anion-exchanger.
 148. The method of claim 142,wherein said VLPs are purified to greater than about 70%.
 149. Themethod of claim 142, wherein the Calicivirus VLPs are Norovirus VLPs.150. A pharmaceutical agent comprising VLPs prepared by a methodaccording to claim
 142. 151. The pharmaceutical agent of claim 150,wherein the pharmaceutical agent comprises a vaccine.